Date of Award
DNA double-strand breaks (DSBs) are dangerous insults to DNA integrity and can lead to genome instability if left unrepaired. However, the immune cell diversification reactions V(D)J recombination and Class Switch Recombination (CSR) require the formation of DSB intermediates, a process that is tightly controlled and strictly limited to developing B and T cells. CSR in B cells diversifies antibodies by joining DSBs between highly repetitive DNA elements, which are separated by 60-200 kb. Switch region DSBs are joined by a mechanism that requires an intact DNA damage response and classical or alternative non-homologous end-joining (CNHEJ and A-NHEJ). Among DNA damage response factors, absence of 53BP1 leads to the most severe defect in CSR. Similarly, the loss of 53BP1 leads to impaired joining of distal DSBs during V(D)J recombination and results in abrogated transchromosomal fusions of dysfunctional telomeres. Interestingly, joining of proximal switch region internal DSBs is not affected by the absence of 53BP1, leading to the hypothesis that 53BP1 affects the joining of only a subset of DSBs. Here I use the ISceI meganuclease system to introduce site-directed DSBs in order to establish the effect of 53BP1 on the joining of trans-chromosomal and intra-chromosomal DSBs separated by various distances. I provide evidence that 53BP1 facilitates joining of intra-chromosomal DSBs, but that this effect is limited to a range that coincides with the spread of DNA damage response factors. I then explore the role of 53BP1 in DNA repair, and find that the absence of 53BP1 results in a distance-independent increase in DNA end resection and that resected DNA is preferentially repaired by microhomology mediated A-NHEJ. Furthermore, analysis of 53BP1 mutants shows that chromatin association, oligomerization, and N-terminal ATM phosphorylation sites are all required for preventing DNA end resection and joining as measured by immunoglobulin CSR. In summary, these data provide new insights the molecular mechanisms by which 53BP1 facilitates DSB joining during CSR and its contribution towards the maintenance of genomic stability.
Bothmer, Anne Helen, "The Role of 53BP1 in DNA Double-Strand Break Repair" (2011). Student Theses and Dissertations. 145.